Studies of the pathogenesis of cystic fibrosis (CF) have highlighted the importance of host defenses at the airway surface in combating microbial infections. The airway epithelial cell plays a key role in pulmonary host defense through a number of different pathways. During the last cycle of this grant, we identified and characterized from humans and mice novel molecules, called beta-defensins, which are secreted by epithelial cells on to the surface of the airway where they contribute to bacterial killing. Mice individually deficient in two mouse beta-defensin genes were created and subjected to preliminary analysis. Recent work by others has determined a family of transmembrane proteins called Toll Like Receptors (TLRs) important signaling innate immunity and initiating acquired immune responses. The goal of this proposal is to evaluate the role of the pulmonary epithelia in initiating the innate immune response in murine models to two pathogens important to the pathogenesis of CF, H. influenzae and P. aeruginosa. In preclinical studies, we document expression of human TLR in airway epithelia cells and its role in signaling innate immunity. Studies in mice challenged with H. influenzae documented the importance of murine beta-defensin 1 in clearing the infection and TLR4 in sensing the infection and signaling an innate immune response. Studies with P. aeruginosa suggest a critical role of TLR4 in the effector response of innate immunity. Specific Aim 1 will evaluate the relative role of the epithelia versus macrophages in sensing and signaling innate immunity and will attempt to confirm/identify specific TLRs involved in detecting the pathogens under study. Specific Aim 2 will characterize the role of mBD- 1 and mBD-3 in responding to intrapulmonary challenge of prototypic pathogens. Specific Aim 3 will delineate the proposed defect(s) in effector cell function to P. aeruginosa observed in TLR deficient mice.